Effect of Bawang Dayak (Eleutherine palmifolia (L) Merr) crude extract towards bacteria

inhibition zone and carp (Cyprinus carpio) hematology

Maftuch

Citation: AIP Conference Proceedings 1844, 020010 (2017); doi: 10.1063/1.4983421

View online: http://dx.doi.org/10.1063/1.4983421
View Table of Contents: http://aip.scitation.org/toc/apc/1844/1
Published by the American Institute of Physics
 Effect of Bawang Dayak (Eleutherine palmifolia (L) Merr)
Crude Extract towards Bacteria Inhibition Zone and Carp
(Cyprinus carpio) Hematology
Maftuch1a)
1
Dept. Aquaculture, Faculty of Fisheries and Marine Science, Brawijaya University, Malang, East Java, Indonesia

a) Corresponding author: maftuch@ub.ac.id

Abstract. Negative impacts of antibiotics and chemical substance usage in aquaculture demand the researchers discover
more efficient alternative yet environmentally friendly to overcome fish diseases. One alternative is by using Bawang
Dayak (Eleutherine palmifolia (L.) Merr). This research aimed to reveal the effect of Bawang Dayak crude extract
towards the inhibition zone of A. hydrophilia, V. harveyi, and P. fluorescens bacteria. Furthermore, it was also conducted
to investigate the carp (C. carpio) hematology which was infected with A. hydrophila bacteria, and find the most
appropriate dose of Bawang Dayak crude extract to inhibit the bacteria. This experimental research was performed by
using Completely Randomized Design with 4 treatments and 3 replications. The best result of the zone of inhibition test
in A. hydrophila bacteria was at the dose of 70 ppm while V. harveyi and P. Fluorescens bacteria were at the dose of 85
ppm. Then, fish hematology was found best at the dose of 80 ppm. Bawang Dayak crude extract was significant towards
the inhibition zone of A. hydrophila, V. harveyi and P. Fluorescens bacteria, and carp hematology which was infected
with A. hydrophila bacteria.

INTRODUCTION
Carp is a freshwater fish which is economically important, and therefore it is widely cultivated. Not only
cultivated in certain ponds, but carp is also cultivated in a rice field along with the crops.1 According to Ardiansyah
et al. (2011)2, the intensification of cultivation should be applied to meet the demand for fisheries products which
increase continuously. However, the intensification of cultivation can cause side effects such as disease development
caused by pathogenic bacteria.
Fish diseases in aquaculture are commonly caused by environmental conditions including poor water quality.
Moreover, an outbreak of diseases generally caused by bacteria. There are several techniques to control fish diseases
such as giving antibiotic substances. However, the use of antibiotic substances causes another problem such as the
spread of antibiotic resistant bacteria and environmental hazard.3-4
Natural resources in Indonesia contain abundant antibiotic contents which have not been fully exploited. Plants
are the common resources that have been explored to be used as antibiotics. Numerous kinds of plants in Indonesia
that have been known to have antibacterial activities are chamber bitter (Phyllanthus niruri), garlic (Allium sativum),
mangrove, and country-almond (T. cattapa L.).5-8 Onion (Eleutherine sp.) is known as the source of antibacterial
agents. In this study, Bawang Dayak (E. palmifolia (L) Merr) is analyzed to know its antibacterial capability and its
effect on carp (Cyprinus carpio) hematology.

The 7th International Conference on Global Resource Conservation

AIP Conf. Proc. 1844, 020010-1–020010-7; doi: 10.1063/1.4983421
Published by AIP Publishing. 978-0-7354-1516-4/$30.00

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 EXPERIMENTAL DETAILS

Extraction
Bawang Dayak (Eleutherine palmifolia Merr.) was obtained from a traditional market near Universitas
Brawijaya. The extraction method was using Chatterjee et al. (2011)9 modification. At the beginning, the sample
was dried and then it was ground by using a blender. The ground Bawang Dayak was weighed and then mixed with
ethanol solvent 96 % in a 1000 ml beaker glass with 1:4 in proportion; it meant every 1 gram of Bawang Dayak was
soaked in 4 ml ethanol. Then it was stored for 1x24 hours (maceration). After maceration, Bawang Dayak was
strained by using smooth filter paper. The filtrate was evaporated by using rotary evaporator at 50 oC until the
ethanol solvent 96% evaporated. Thus, it produced crude extract in a paste form.

Phytochemical Analysis.
Phytochemical analysis was performed by following Harborne method (1984)10. It was used to analyze
flavonoid, tannin, saponin and alkaloid.

Minimum Inhibitory Concentration (MIC).

The MIC test of Bawang Dayak crude extract was performed by using a method from (Dhayanithi, et al.,
2010)11, with slight modification. The MIC were determined by diluting the Bawang Dayak extract with various
concentrations (0; 0.01; 0.1; 1; 10; 100; and 1000 ppm). Specifically, 0.1 ml of standardized inoculums of tested
bacteria (A. hydrophila, V. harveyi dan P. fluorescens ) 10 X 109 CFU/ml was added to each tube. The tubes were
incubated at 30 ºC for 24 hours. MIC was determined as the lowest concentration of the extracts permitting no
visible growth (no turbidity). The inhibition zone was also investigated in all varied doses of Bawang Dayak crude
extract. Different doses of 100 μl of Bawang Dayak crude extracts were loaded in each well. After 24 hours at 30 ºC,
the clear zone was measured.

Carp Infection Caused by Bacteria.

C. carpio juvenile fish was obtained from Freshwater fish Brood stock Center (BBAT) Punten, there were 270
fish in a size of 7 – 12 cm. Each aquarium was filled with 15 carps and it was acclimatized for 3 days. Infecting
bacteria to fish sample was done by soaking the fish in a tank filled with water which has been added 10 7 cell/ml
bacteria for 24 hours. Then, the fish was moved into a tank containing fresh water. Its clinical symptoms (pale body
color, flaky scales and often swimming to the surface) were observed. The fish was captivated for 1 week. Then, the
water quality including temperature, DO, and pH was measured at 06.00 a.m and 06.00 p.m.

Bawang Dayak crude extract treatment.

The crude extract of Bawang Dayak was put in an aquarium filled with 12 liters of water by using immersion
method.12 Then, the crude extract was added based on defined doses, 50 ppm, 60 ppm, 70 ppm, and 80 ppm. The
aquarium was equipped with aeration to supply oxygen. Each aquarium was filled with 15 carps. The soaking
process was performed until the fish showed erratic manner, in this case, 11 hours. Then, the carp was moved in a
new aquarium filled with freshwater. Next, the blood sample was taken to measure its total erythrocyte, leukocyte,
and hemoglobin. The blood sample was taken every 12 hours with 4 times blood taking.

Hematology Analysis
Blood Sample Taking

Carp blood was taken on its caudal peduncle part by using a syringe containing Na Citrate 3.8 %. The
syringe was injected with the needle at 45o position and it was pulled slowly until the blood coming inside the
syringe. Then, the blood sample was put in an Eppendorf tube.

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 Total Erythrocyte Count

According to Mones (2008)13, red blood cell count can be performed by using hemocytometer. Red blood cell
could be counted by using this following formula:
ܵ‫ ܯܦ‬ൌ  ሺܽȀ݊ሻ‫ݔ‬ሺͳȀ‫ݒ‬ሻ‫݌ܨݔ‬
(1)
Before being counted, the blood sample mixed with anticoagulant by using Thoma erythrocyte pipette up to 0.5
in scale. Then, Hayem’s solution was also added until the scale reached 101 (dilution 1:200). The pipette was gently
shaken to mix the blood and Hayem’s solution completely. After that, 4 initial drops were discarded and the next
drop was put in hemocytometer; it was done based on the prediction that the fifth drop contained a complete mixture
of blood and Hayem’s solution. Then, hemocytometer was covered with cover glass. The blood specimens were
observed using a microscope (M=400x). The total erythrocyte was counted in 5 big grids found in cell counting
chamber.

Total Leukocyte Count

In white blood cell count, According to Mones (2008)13, the first thing that had to do was sucking the blood
sample mixed with anticoagulant by using Thoma leukocyte pipette up to 0.5 in scale. Then, Turk’s solution was
also sucked until the scale showing 11 (dilution 1:20). The pipette was gently shaken to mix the blood and Turk’s
solution completely. After that, 4 initial drops were discarded and the next drop was put in hemocytometer; it was
done based on the prediction that the fifth drop contained a complete mixture of blood and Turk’s solution. Then,
hemocytometer was covered with cover glass. The observation using microscope was done with 400x magnification.
The total leukocyte was counted in 4 big grids found in cell counting chamber.

Hemoglobin Level Count

Hemoglobin level count was done by using Sahli method and based on Sebastio (2011)14 procedure, the first
step was filling the Sahli tube with 0.1 N HCL solution. Then, 0.02 fish blood was taken and moved into Sahli tube
containing 0.1 N HCL solution. Next, the tube was gently shaken and let rest for 5 minutes. After that, purified
water was added to the Sahli tube so the sample color was similar to the standard color of Sahli tube.

RESULT AND DISCUSSION

Phytochemical Test.
The phytochemical test was done to know the secondary metabolite in the plant including flavonoid, tannin,
saponin and alkaloid. The result of phytochemical test could be seen in Table 1.
TABLE 1. Phytochemical Test Result of Bawang Dayak (E. Palmifolia (L) Merr)10.

Fitokimia Test Test Result Note

Flavonoid + Red Color/Light Red
Saponin + Formed permanent foam
Tanin Tanin Galat + Ink blue / black
Tanin Katekol + There is red sludge
Alkaloid Mayer reagent - There is not white sludge
Dragendrof reagent - There is not orange sludge

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 MIC Test.
MIC (Minimum Inhibitory Concentration) test used different doses of Bawang Dayak (E. palmifolia L Merr )
crude extract. Differentiating the doses was aimed at knowing the smallest dose to inhibit or kill A. hydrophila, P.
fluorescens, and V. harveyi bacteria. The results of MIC test showed differences in each treatment after being
observed by using spectrophotometer as shown in Table 2.
TABLE 2. MIC Test Result by Using Spectrophotometer

No. Bacteri Consentration of Absorbance Color

extract (ppm)
1000 0.222 Clear
100 0.159 Clear
Aeromonas

10 0.142 Clear
hydrophila

1 0.164 Cloudy
1 0.1 0.175 Cloudy
0.01 0.180 Cloudy
0 0.168 Cloudy
Control (+) 0.145 Clear
Control (-) 1.281 Cloudy
1000 0.116 Clear
100 0.114 Clear
Pseudomonas

10 0.111 Clear
fluoresens

1 0.122 Clear
2 0.1 0.107 Clear
0.01 0.116 Cloudy
0 0.294 Cloudy
Control (+) 0.098 Clear
Control (-) 0.906 Cloudy
1000 0.332 Clear
100 0.209 Clear
Vibrio harveyi

10 0.185 Clear
1 0.205 Cloudy
3 0.1 0.214 Cloudy
0.01 0.226 Cloudy
0 0.197 Cloudy
Control (+) 0.105 Clear
Control (-) 1.399 Cloudy

Based on the MIC test result by using a spectrophotometer, it could be seen that concentration 10 ppm with the
positive control (+) produced a clear solution. It indicated that the dose of 10 ppm could inhibit the bacteria. In
positive control (+), there was only Bawang Dayak (E. palmifolia (L) Merr) crude extract with 1000 ppm as the
highest dose without bacteria addition. The absorbance value in 10 ppm of Bawang Dayak (E. palmifolia (L) Merr)
crude extract was 0.142, in A. hydrophila bacteria was close to positive control with 0.145 absorbance value. Then,
the absorbance value of P. fluorescens bacteria in 0.1 ppm concentration was 0.107, close to positive control with
0.098 absorbance value. The absorbance value of V. harveyi in 10 ppm concentration was 0.185, close to positive
control with 0.105 absorbance value.
The results of MIC showed that P. fluorescens bacteria more sensitive then A. hydrophila and V. harveyi.
Extracts of Bawang Dayak (E. palmifolia (L) Merr) by using ethanol showed good inhibitory activity with low MIC
concentration (0.1-1000 ppm) (Table. 2). Therefore, MIC results can indirectly predict the concentration of extract
which should be used as minimum dosage. Ethanol has higher polarity and hence the antibacterial compounds are
polar compounds which are under purification process.11 Bawang Dayak (E. palmifolia (L) Merr) is a one of the
easily available some like onion by applying this concentration extract will significantly reduce the bacterial
population in fishes and reduce infection of fishes.

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 Hematology Observation
Total Erythrocyte.

The total erythrocyte based on observation during cultivation could be seen in Table 5. This research used 4
treatments consisting of A= 50 ppm, B= 60 ppm, C= 70 ppm and D= 80 ppm with 3 replications. The observation
was done 4 times every 12 hours. The total erythrocyte count could be seen in Fig.1.

FIGURE 1. Mean of Total Erythrocyte (x106 cell/mm3) in Carp

Treatment D with the dose of 80 ppm was the best treatment because the total value of erythrocyte was back to
normal after 36 hours of observation, it was 2.29 x 106 cells/mm3. The total of erythrocytes had approached the
average value of negative control fish. The total of fish erythrocytes in negative control during observation was 2.2 x
106 cell / mm3. This results corresponding to Hrubec, et al., (2000)15 that normal interval RBC (X 106/μL) in
Tilapia fish was 1.91-2.83. The total of erythrocytes in treatment D was the highest compared to treatment A with
the dose of 50 ppm which showed the lowest result, it was because the fish suffered from anemia. Anemia decreased
the body metabolism rate and led to a lack of energy which made the fish weak and had no appetite. Erythrocytes in
the body play an important role in metabolic processes, in which the erythrocytes serves to transfer oxygen
throughout the body. The reduced number of erythrocytes lowers the oxygen supply to the tissues and organs of the
body, and declines the food supply to the cells.

Total Leukocyte

The total leukocyte based on observation during cultivation could be seen in Figure 2. Treatment D with the dose
of 80 ppm was the best treatment because the total value of leukocyte was normal, it was 8.54 x 103 cells/mm3.
Then, the total of leucocytes had approached the negative control fish with 8.4 x 10 3 cells/mm3. According to
Hrubec, et al., (2000)15, that normal interval of WBC (/μL) was 21,559-154,690. So, the result was lower than the
normal condition. This condition was meaning that the dose and duration of treatment extract could be the recovery
of infection of fish. The leucocytes of infected fish would increase, otherwise, the leucocyte of healthy fish would
decrease. Leucocytes were the defense in fish that would quickly respond to the presence of pathogens in the body.
When the disease attacked, leukocytes would perform phagocytosis to the substances causing it.

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 FIGURE 2. Mean of Total Leukocyte (x103 cell/mm3) in Carp

Hemoglobin.

The number of hemoglobin based on observation during cultivation could be seen in Fi.3. Treatment D at a dose
of 80 ppm contained the highest number of hemoglobin compared to treatment A at a dose of 50 ppm,that was
because the fish was still in recovery. The total hemoglobin of fish which suffered from stress and attacked by
bacteria decreased. That was because the fish suffered from anemia. The dose that gave the best result was found in
treatment D at a dose of 80 ppm. It was because after 36 hours fish hemoglobin had returned to its normal state by 7
g/dL and it was in accordance with the observation on the negative control fish. According to Hrubec, et al.,
(2000)15, that normal interval of Hemoglobin (g/dL) was 7.0-9.8. That was mean after Giving Bawang Dayak crude
extract at a dose of 80 ppm was able to restore hemoglobin to a normal amount and inhibit the growth of A.
hydrophila bacteria.

FIGURE 3. Total Mean of Hemoglobin (g %) in Carp During Cultivation

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 CONCLUSION
The crude extract of Bawang Dayak contained various kinds of phytochemicals which had antimicrobial
activities. Thus it could inhibit the growth of A. hydrophila, V. harveyi and P. fluorescens bacteria. Based on the
hematology observation, it is suggested that 80 ppm of Bawang Dayak crude extract is the best concentration to treat
microbes and it has no significant effect on carp (C. carpio) blood.

ACKNOWLEDGEMENTS
We would like to thank Dr. Widodo, Prof. Muhaimin Rifai, Heny Suprastyani for his technical assistance and
publications. And also Dwi Retno Fatmawati, Riska Rinaldi, Ika Khairatun Nisyak, Dimas Ardiansyah participated
in the research design and workmanship in the laboratory. Asep Awaludin Prihanto involved in the preparation of
the manuscript. All authors have read and approved the final manuscript.

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